Treatment of diseases

ABSTRACT

The invention provides (i) a method of treating metabolic syndrome in an animal, (ii) a method of suppressing the appetite of an animal, (iii) a method of treating obesity in an animal, (iv) a method of reducing the weight of an animal in need thereof, (v) a method of reducing a blood lipid level in an animal in need thereof, (vi) a method of treating non-alcoholic steatohepatitis in an animal, and (vii) a method of inhibiting adipogenesis. The methods comprise administering an effective amount of an active ingredient, wherein the active ingredient comprises a diketopiperazine, a prodrug of a diketopiperazine or a pharmaceutically-acceptable salt of either of them to the animal. The invention also provides a kit comprising a container holding a diketopiperazine, a prodrug of a diketopiperazine or a pharmaceutically-acceptable salt of either of them; and instructions for administration of the diketopiperazine, the prodrug or the pharmaceutically-acceptable salt according to a method of the invention. The diketopiperazines have the formula given in the application.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/512,151, filed Jul. 27, 2011, and U.S.Provisional patent application Ser. No. 61/380,426, filed Sep. 7, 2010,the complete disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a treatment for metabolic syndrome comprisingadministering to an animal in need thereof an effective amount of adiketopiperazine of formula I set forth below, a prodrug of adiketopiperazine of formula I or a pharmaceutically-acceptable salt ofeither of them. The invention further relates to a method of suppressingthe appetite of an animal, a method of treating obesity in an animal, amethod of reducing the weight of an animal in need thereof, a method ofreducing a blood lipid level in an animal in need thereof, a method oftreating non-alcoholic steatohepatitis in an animal, and a method ofinhibiting adipogenesis in an animal. These methods also compriseadministering an effective amount of a diketopiperazine of formula I, aprodrug of a diketopiperazine of formula I or apharmaceutically-acceptable salt of either of them to the animal. Theinvention also relates to a kit comprising a container holding adiketopiperazine of formula I, a prodrug of a diketopiperazine offormula I or a pharmaceutically-acceptable salt of either of them; andinstructions for administration of the diketopiperazine, the prodrug orthe pharmaceutically-acceptable salt according to a method of theinvention.

BACKGROUND

Metabolic syndrome is a complex of risk factors for cardiovasculardisease and type 2 diabetes. The most widely recognized of the riskfactors are atherogenic dyslipidemia, elevated blood pressure andelevated plasma glucose. In addition, patients with thesecharacteristics commonly manifest a prothrombotic state and aproinflammatory state. Atherogenic dyslipidemia consists of anaggregation of lipoprotein abnormalities that includes elevated serumtriglyceride and apolipoprotein B, increased small low-densitylipoprotein particles and a reduced level of high-density lipoproteincholesterol. Most patients with metabolic syndrome also have abdominal(central) obesity and insulin resistance. For general background onmetabolic syndrome factors and diagnosis, see Alberti et al.,Circulation, 120:1640-1645 (2009).

Metabolic syndrome is common and has a rising prevalence worldwide,which relates largely to increasing obesity and sedentary lifestyles.Patients with the metabolic syndrome are at twice the risk of developingcardiovascular disease over the next five to ten years as individualswithout the syndrome. The risk over a lifetime undoubtedly is evenhigher. Furthermore, the metabolic syndrome confers a five-fold increasein risk for type 2 diabetes mellitus. Metabolic syndrome is now both apublic health and a clinical problem, and there is clearly a need fornew and effective treatments for metabolic syndrome.

SUMMARY OF THE INVENTION

The invention provides such a treatment. In particular, the inventionprovides a method of treating metabolic syndrome in an animal. Themethod comprises administering to the animal an effective amount of anactive ingredient, wherein the active ingredient comprises adiketopiperazine of formula I given below, a prodrug of adiketopiperazine of formula I or a pharmaceutically-acceptable salt ofeither of them.

The invention also provides a method of suppressing the appetite of ananimal, a method of treating obesity in an animal, a method of reducingthe weight of an animal in need thereof, a method of reducing a bloodlipid level in an animal in need thereof, a method of treatingnon-alcoholic steatohepatitis in an animal, and a method of inhibitingadipogenesis in an animal.

These methods also comprise administering an effective amount of anactive ingredient, wherein the active ingredient comprises adiketopiperazine of formula I, a prodrug of a diketopiperazine offormula I or a pharmaceutically-acceptable salt of either of them to theanimal.

The invention further provides a kit comprising a container holding adiketopiperazine of formula I, a prodrug of a diketopiperazine offormula I or a pharmaceutically-acceptable salt of either of them; andinstructions for administration of the diketopiperazine, the prodrug orthe pharmaceutically-acceptable salt according to a method of theinvention.

The diketopiperazines useful in the invention have the following formulaI:

wherein:

R¹ and R², which may be the same or different, each is:

-   -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, aspartic        acid, asparagine, glutamic acid, glutamine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine, cysteine, homocysteine, methionine, penicillamine or        ornithine; or    -   (b) R¹ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline        and/or R² is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline; or    -   (b) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —COOH group replaced by a —COOR³ group, wherein each            R³ may independently be a substituted or unsubstituted            alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,            arylalkyl or heteroaryl;        -   (iv) a —COOH group replaced by a —CON(R⁴)₂ group, wherein            each R⁴ may independently be H or a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (v) an —SH group replaced by —S—S—CH₂—CH(NH₂)—COOH or            —S—S—CH₂—CH₂—CH(NH₂)—COOH;        -   (vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (vii) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH            group; and/or        -   (viii) an H which is attached to a carbon atom replaced by a            halogen;        -   provided, however, that when R¹ is the side chain of            histidine or a derivative of the side chain of histidine,            then R² cannot be proline or hydroxyproline, and that when            R² is the side chain of histidine or a derivative of the            side chain of histidine, then R¹ cannot be proline or            hydroxyproline.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Effect of DA-DKP on adipogenesis. Statistical significanceversus untreated controls is indicated by an asterisk (*).

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS OF THEINVENTION

The invention provides (i) a method of treating metabolic syndrome in ananimal, (ii) a method of suppressing the appetite of an animal, (iii) amethod of treating obesity in an animal, (iv) a method of reducing theweight of an animal in need thereof, such as an obese or overweightanimal, (v) a method of reducing a blood lipid level in an animal inneed thereof, and (vi) a method of treating non-alcoholicsteatohepatitis in an animal.

The methods of the invention comprise administering an effective amountof an active ingredient, wherein the active ingredient comprises adiketopiperazine, a prodrug of a diketopiperazine or apharmaceutically-acceptable salt of either of them to the animal,wherein the diketopiperazine has the following formula I:

wherein:

R¹ and R², which may be the same or different, each is:

-   -   (a) a side chain of an amino acid, wherein the amino acid is        glycine, alanine, valine, norvaline, α-aminoisobutyric acid,        2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine,        isoleucine, norleucine, serine, homoserine, threonine, aspartic        acid, asparagine, glutamic acid, glutamine, lysine,        hydroxylysine, histidine, arginine, homoarginine, citrulline,        phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,        thyroxine, cysteine, homocysteine, methionine, penicillamine or        ornithine; or    -   (b) R¹ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline        and/or R² is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with        the adjacent ring nitrogen forms proline or hydroxyproline; or    -   (b) a derivative of a side chain of an amino acid, wherein the        amino acid is one of those recited in (a), and the derivatized        side chain has:        -   (i) an —NH₂ group replaced by an —NHR³ or —N(R³)₂ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (ii) an —OH group replaced by an —O—PO₃H₂ or —OR³ group,            wherein each R³ may independently be a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (iii) a —COOH group replaced by a —COOR³ group, wherein each            R³ may independently be a substituted or unsubstituted            alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,            arylalkyl or heteroaryl;        -   (iv) a —COOH group replaced by a —CON(R⁴)₂ group, wherein            each R⁴ may independently be H or a substituted or            unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,            alkylaryl, arylalkyl or heteroaryl;        -   (v) an —SH group replaced by —S—S—CH₂—CH(NH₂)—COOH or            —S—S—CH₂—CH₂—CH(NH₂)—COOH;        -   (vi) a —CH₂— group replaced by a —CH(NH₂)— or a —CH(OH)—            group;        -   (vii) a —CH₃ group replaced by a —CH₂—NH₂ or a —CH₂—OH            group; and/or        -   (viii) an H which is attached to a carbon atom replaced by a            halogen;        -   provided, however, that when R¹ is the side chain of            histidine or a derivative of the side chain of histidine,            then R² cannot be proline or hydroxyproline, and that when            R² is the side chain of histidine or a derivative of the            side chain of histidine, then R¹ cannot be proline or            hydroxyproline.

By “replaced” is meant that, with reference to the formula of an aminoacid side chain, the specified group is replaced by the other specifiedgroup. For instance, the formula of the isoleucine side chain is—CH(CH₃)—CH₂—CH₃. If the terminal —CH₃ group is replaced with a —CH₂—OHgroup, then the formula of the resulting derivatized isoleucine sidechain would be —CH(CH₃)—CH₂—CH₂—OH. As another example, the formula ofthe alanine side chain is —CH₃. If one of the hydrogen atoms is replacedby a chlorine atom, then the resulting derivatized alanine side chainwould be —CH₂—Cl. Note that the side chain of glycine is —H and, if thisH is replaced by a chlorine (or other halogen) atom, the resulting sidechain will —Cl, with the chlorine atom attached to the ring carbon(e.g., R¹=—Cl)

By “side chain” of an amino acid is meant that portion of the amino acidattached to the common

backbone of all of the amino acids listed above. For instance, the sidechain of glycine is —H, the side chain of alanine is —CH₃, and the sidechain of serine is —CH₂OH.

By “hydrophobic” is meant a side chain or side chain derivative that isuncharged at physiological pH and is repelled by an aqueous solution.

By “neutral” is meant a side chain or side chain derivative that isuncharged at physiological pH.

By “basic” is meant a side chain or side chain derivative that ispositively charged at physiological pH.

By “acidic” is meant a side chain or side chain derivative that isnegatively charged at physiological pH.

By “alkyl” is meant a saturated straight-chain or branched hydrocarboncontaining 1-10 carbon atoms, preferably 1-6, carbon atoms. “Loweralkyl” means a saturated straight-chain or branched hydrocarboncontaining 1-6 carbon atoms.

By “cycloalkyl” is meant a saturated cyclic hydrocarbon containing atleast one ring, each ring containing at least three carbon atoms.Preferably, the cycloalkyl contains one ring of 4-8 carbon atoms.

By “heterocycloalkyl” is meant a cycloalkyl having one or more of thering carbon atoms of at least one of the rings replaced by an O, Sand/or N.

By “aryl” is meant an aromatic group having at least one aromatic ring(e.g., phenyl).

By “alkylaryl” is meant a lower alkyl having an H replaced by an aryl(e.g., —CH₂—C₆H₅ or —CH₃CH(C₆H₅)CH₃).

By “arylalkyl” is meant an aryl having an H replaced by a lower alkyl(e.g., —C₆H₄—CH₃).

By “heteroaryl” is meant an aryl having one or more of the ring carbonatoms of at least one of the rings replaced by an O, S and/or N.

By “substituted” is meant that the moiety is substituted with one ormore substituents selected from the following group: —OH, NH₂, —SH,—COOH and/or a halogen atom.

By “halogen” is meant chlorine, fluorine, bromine or iodine. Preferredis chlorine or bromine.

Most preferred for use in the invention are diketopiperazines of formulaI wherein R¹, R² or both is the side chain of aspartic acid or glutamicacid or a derivative of such a side chain wherein the —COOH group isreplaced by a —COOR³ group or a —CON(R⁴)₂ group, wherein R³ and R⁴ aredefined above. Of this group of compounds, preferred arediketopiperazines comprising the side chains of aspartic acid andalanine (Asp-Ala DKP or DA-DKP), the side chains of glutamic acid andalanine (Glu-Ala DKP or EA-DKP), the side chains of tyrosine andaspartic acid (Tyr-Asp DKP or YD-DKP), the side chains of tyrosine andglutamic acid (Tyr-Glu DKP or YE-DKP) and derivatives of the asparticacid or glutamic acid side chains of these four diketopiperazineswherein the —COOH group is replaced by a —COOR³ group or a —CON(R⁴)₂group, wherein R³ and R⁴ are defined above. Most preferred is DA-DKP.

Also preferred are diketopiperazines wherein R¹, R² or both arehydrophobic side chains or hydrophobic side chain derivatives. By“hydrophobic side chain derivative” is meant that the derivatized sidechain is hydrophobic. In particular, preferred are diketopiperzineswherein R¹ and/or R², which may be the same or different, each is theside chain of glycine, alanine, valine, norvaline, α-aminobutyric acid,leucine, isoleucine, norleucine, methionine, phenylalanine, tryptophanor tyrosine. Of this group, preferred are diketopiperzines wherein R¹and/or R², which may be the same or different, each is the side chain ofglycine, alanine, valine, norvaline, α-aminobutyric acid, leucine,isoleucine, norleucine, methionine or tyrosine, more preferably alanine,valine, norvaline, α-aminobutyric acid, leucine, isoleucine ornorleucine. Generally, proline is not preferred, although it ishydrophobic.

Additional preferred diketopiperazines are those wherein R¹, R² or bothside chains are neutral side chains or neutral side chain derivatives.By “neutral side chain derivative” is meant that the derivatized sidechain is neutral. In particular, preferred are diketopiperzines whereinR¹ and/or R², which may be the same or different, each is the side chainof asparagine, glutamine, serine, homoserine, threonine, tyrosine,cysteine or methionine. Of this group, preferred are diketopiperzineswherein R¹ and/or R², which may be the same or different, each is theside chain of asparagine, glutamine, serine or threonine.

Also preferred are diketopiperazines wherein R¹, R² or both are basicside chains or basic side chain derivatives. By “basic side chainderivative” is meant that the derivatized side chain is basic. Inparticular, preferred are diketopiperzines wherein R¹ and/or R², whichmay be the same or different, each is the side chain of citrulline,2,4-diaminobutryic acid, 2,3-diaminobutyric acid, lysine, hydroxylysine,arginine, homoarginine, citrulline, p-aminophenylalanine, or ornithine.Of this group, preferred are diketopiperzines wherein R¹ and/or R²,which may be the same or different, each is the side chain ofcitrulline, 2,4-diaminobutryic acid, 2,3-diaminobutyric acid, lysine,arginine, homoarginine or p-aminophenylalanine. Generally, histidine isnot preferred, although it is basic.

Further preferred diketopiperazines are those wherein R¹, R² or both isthe side chain of methionine, the side chain of arginine or a derivativeof these side chains. Most preferred of this group is a diketopiperazinewherein R¹ is the side chain of methionine and R² is the side chain ofarginine (Met-Arg DKP or MR-DKP).

Highly preferred for use herein is DA-DKP. Patients with metabolicsyndrome can be treated by administering an effective amount of aDA-DKP, a prodrug of a DA-DKP or a pharmaceutically-acceptable salt ofeither of them. DA-DKP has multiple activities that should make it aparticularly effective treatment for metabolic syndrome.

First, DA-DKP has been found to suppress the appetite. DA-DKP has alsobeen found to inhibit the differentiation of preadipocytes intoadipocytes. Accordingly, it is expected to be beneficial in thetreatment of metabolic syndrome by contributing to weight loss and thetreatment of obesity. It can also be used as a treatment for obesity andfor weight loss in general, not just the obesity and excess weightassociated with metabolic syndrome.

In addition, DA-DKP has also been found to reduce levels of lipids(cholesterol, triglycerides, low-density lipoprotein and high-densitylipoprotein) in blood. For this reason, it is expected to havebeneficial effects on the dyslipidemia which is one of the risk factorsof metabolic syndrome.

Another pathological condition associated with the metabolic syndrome isthe development of non-alcoholic steatohepatitis (NASH) in obesepatients. NASH is characterized by an accumulation of lipid molecules inthe liver accompanied by severe inflammation, fibrosis, andinsulin/leptin resistance. In view of the foregoing activities ofDA-DKP, it is expected to be beneficial in the treatment of NASH,whether associated with metabolic syndrome or independent of metabolicsyndrome.

Further, DA-DKP has beneficial effects on the vascular endothelialbarrier, including the ability to inhibit vascular hyperpermeability.Accordingly, it is expected to be an effective treatment for thoseexhibiting early signs of, or with a predisposition to, type 2 diabetes,such as those diagnosed with metabolic syndrome, to reduce, delay oreven potentially prevent the vascular complications of diabetes. SeeApplicant's co-pending provisional application No. 61/380,404, filedSep. 7, 2010.

Finally, DA-DKP is known to inhibit the aggregation of platelets, to beanti-inflammatory and to inhibit the activation of T-cells. See U.S.Pat. Nos. 6,555,543 and 7,732,403. Accordingly DA-DKP is expected to beuseful in combating the prothrombotic and proinflammatory statesobserved in metabolic syndrome.

Methods of making diketopiperazines are well known in the art, and thesemethods may be employed to synthesize the diketopiperazines of theinvention. See, e.g., U.S. Pat. Nos. 4,694,081, 5,817,751, 5,990,112,5,932,579 and 6,555,543, US Patent Application Publication Number2004/0024180, PCT applications WO 96/00391 and WO 97/48685, and Smith etal., Bioorg. Med. Chem. Letters, 8, 2369-2374 (1998), the completedisclosures of which are incorporated herein by reference.

For instance, diketopiperazines can be prepared by first synthesizingdipeptides. The dipeptides can be synthesized by methods well known inthe art using L-amino acids, D-amino acids or a combination of D- andL-amino acids. Preferred are solid-phase peptide synthetic methods. Ofcourse, dipeptides are also available commercially from numeroussources, including Sigma-Aldrich, St. Louis, Mo. (primarily customsynthesis), Phoenix Pharmaceuticals, Inc., Belmont, Calif. (customsynthesis), Fisher Scientific (custom synthesis) and Advanced ChemTech,Louisville, Ky.

Once the dipeptide is synthesized or purchased, it is cyclized to form adiketopiperazine. This can be accomplished by a variety of techniques.

For example, U.S. Patent Application Publication Number 2004/0024180describes a method of cyclizing dipeptides. Briefly, the dipeptide isheated in an organic solvent while removing water by distillation.Preferably, the organic solvent is a low-boiling azeotrope with water,such as acetonitrile, allyl alcohol, benzene, benzyl alcohol, n-butanol,2-butanol, t-butanol, acetic acid butylester, carbon tetrachloride,chlorobenzene chloroform, cyclohexane, 1,2-dichlorethane, diethylacetal,dimethylacetal, acetic acid ethylester, heptane, methylisobutylketone,3-pentanol, toluene and xylene. The temperature depends on the reactionspeed at which the cyclization takes place and on the type ofazeotroping agent used. The reaction is preferably carried out at50-200° C., more preferably 80-150° C. The pH range in which cyclizationtakes place can be easily determine by the person skilled in the art. Itwill advantageously be 2-9, preferably 3-7.

When one or both of the amino acids of the dipeptide has, or isderivatized to have, a carboxyl group on its side chain (e.g., asparticacid or glutamic acid), the dipeptide is preferably cyclized asdescribed in U.S. Pat. No. 6,555,543. Briefly, the dipeptide, with theside-chain carboxyl still protected, is heated under neutral conditions.Typically, the dipeptide will be heated at from about 80° C. to about180° C., preferably at about 120° C. The solvent will be a neutralsolvent. For instance, the solvent may comprise an alcohol (such asbutanol, methanol, ethanol, and higher alcohols, but not phenol) and anazeotropic co-solvent (such as toluene, benzene, or xylene). Preferably,the alcohol is butan-2-ol, and the azeotropic co-solvent is toluene. Theheating is continued until the reaction is complete, and such times canbe determined empirically. Typically, the dipeptide will be cyclized byrefluxing it for about 8-24 hours, preferably about 18 hours. Finally,the protecting group is removed from the diketopiperazine. In doing so,the use of strong acids (mineral acids, such as sulfuric or hydrochloricacids), strong bases (alkaline bases, such as potassium hydroxide orsodium hydroxide), and strong reducing agents (e.g., lithium aluminumhydride) should be avoided, in order to maintain the chirality of thefinal compound.

Dipeptides made on solid phase resins can be cyclized and released fromthe resin in one step. See, e.g., U.S. Pat. No. 5,817,751. For instance,the resin having an N-alkylated dipeptide attached is suspended intoluene or toluene/ethanol in the presence of acetic acid (e.g., 1%) ortriethylamine (e.g., 4%). Typically, basic cyclization conditions arepreferred for their faster cyclization times.

To prepare diketopiperazines wherein the amino acid side chains arederivatized, amino acid derivatives can be used in the synthesis of thedipeptides, the dipeptides can be derivatized and/or thediketopiperazines can be derivatized, as is known in the art. See, e.g.,those references cited above.

Other methods of cyclizing dipeptides and of making diketopiperazinesare known in the art and can be used in the preparation ofdiketopiperazines useful in the practice of the invention. See, e.g.,those references listed above. In addition, many diketopiperazinessuitable for use in the present invention can be made from proteins andpeptides as described in U.S. Pat. No. 7,732,403, the completedisclosure of which is incorporated herein by reference. Further,diketopiperazines for use in the practice of the invention can beobtained commercially from, e.g., Syngene, India and HemmoPharmaceuticals Pvt. Ltd, India (both custom synthesis).

The diketopiperazines of the invention include all possiblestereoisomers than can be obtained by varying the configuration of theindividual chiral centers, axes or surfaces. In other words, thediketopiperazines include all possible diastereomers, as well as alloptical isomers (enantiomers).

“Prodrug” means any compound which releases an active parent drug (adiketopiperazine in this case) in vivo when such prodrug is administeredto an animal. Prodrugs of diketopiperazines include derivatives that maybe cleaved in vivo to generate the diketopiperazine. Examples ofdiketopiperazine prodrugs include esters.

The physiologically-acceptable salts of the diketopiperazines andprodrugs of the invention may also be used in the practice of theinvention. Physiologically-acceptable salts include conventionalnon-toxic salts, such as salts derived from inorganic acids (such ashydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the like),organic acids (such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, glutamic, aspartic, benzoic, salicylic,oxalic, ascorbic acid, and the like) or bases (such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cationor organic cations derived from N,N-dibenzylethylenediamine,D-glucosamine, or ethylenediamine). The salts are prepared in aconventional manner, e.g., by neutralizing the free base form of thecompound with an acid.

As noted above, a diketopiperazine of formula I, a prodrug of adiketopiperazine of formula I or a pharmaceutically-acceptable salt ofeither one of them can be used in the methods of the invention. To doso, the diketopiperazine, prodrug or pharmaceutically-acceptable salt isadministered to an animal in need of treatment. Preferably, the animalis a mammal, such as a rabbit, goat, dog, cat, horse or human. Mostpreferably, the animal is a human.

A diketopiperazine of formula I, a prodrug of a diketopiperazine offormula I or a pharmaceutically-acceptable salt of either one of them isused in the present invention as an active ingredient. “Activeingredient” is used herein to mean a compound having therapeutic,pharmaceutical or pharmacological activity, and particularly thetherapeutic, pharmaceutical or pharmacological activities describedherein. The diketopiperazine, prodrug or salt of either one of them isnot used in the present invention as a carrier or as part of a carriersystem of a pharmaceutical composition as described in, e.g., U.S. Pat.Nos. 5,976,569, 6,099,856, 7,276,534 and PCT applications WO 96/10396,WO 2006/023943, WO 2007/098500, WO 2007/121411 and WO 2010/102148.

Effective dosage forms, modes of administration and dosage amounts forthe compounds of the invention (i.e., a diketopiperazine of formula I, aprodrug of a diketopiperazine of formula I or apharmaceutically-acceptable salt of either one of them) may bedetermined empirically using the guidance provided herein. It isunderstood by those skilled in the art that the dosage amount will varywith the particular disease or condition to be treated, the severity ofthe disease or condition, the route(s) of administration, the durationof the treatment, the identity of any other drugs being administered tothe animal, the age, size and species of the animal, and like factorsknown in the medical and veterinary arts. In general, a suitable dailydose of a compound of the present invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. However, the daily dosage will be determined by an attendingphysician or veterinarian within the scope of sound medical judgment. Ifdesired, the effective daily dose may be administered as two, three,four, five, six or more sub-doses, administered separately atappropriate intervals throughout the day. Administration of the compoundshould be continued until an acceptable response is achieved.

In particular, an effective dosage amount of a diketopiperazine offormula I will be from 10 ng/kg/day to 225 mg/kg/day, preferably from500 ng/kg/day to 150 mg/kg/day, most preferably from 1 mg/kg/day to 30mg/kg/day. When given orally to an adult human, the dose will preferablybe from about 1 mg/day to about 10 g/day, more preferably the dose willbe from about 60 mg/day to about 6 g/day, most preferably the dose willbe from about 100 mg/day to about 1200 mg/day, preferably given inseveral doses.

The compounds of the present invention (i.e., a diketopiperazine offormula I, prodrugs thereof and pharmaceutically-acceptable salts ofeither of them) may be administered to an animal patient for therapy byany suitable route of administration, including orally, nasally,parenterally (e.g., intravenously, intraperitoneally, subcutaneously orintramuscularly), transdermally, intraocularly and topically (includingbuccally and sublingually). Generally preferred is oral administrationfor any disease or condition treatable according to the invention.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise a compound or compounds of theinvention as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother compounds, drugs or other materials. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the animal.Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient (i.e., a diketopiperazine of formula I, a prodrug of adiketopiperazine of formula I, a pharmaceutically-acceptable salt ofeither one of them, or combinations of the foregoing) is mixed with oneor more pharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monosterate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may be employedas fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active ingredient, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The invention also provides pharmaceutical products suitable fortreatment of the eye. Such pharmaceutical products includepharmaceutical compositions, devices and implants (which may becompositions or devices).

Pharmaceutical formulations (compositions) for intraocular injection ofa compound or compounds of the invention into the eyeball includesolutions, emulsions, suspensions, particles, capsules, microspheres,liposomes, matrices, etc. See, e.g., U.S. Pat. No. 6,060,463, U.S.Patent Application Publication No. 2005/0101582, and PCT application WO2004/043480, the complete disclosures of which are incorporated hereinby reference. For instance, a pharmaceutical formulation for intraocularinjection may comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, suspensions or emulsions,which may contain antioxidants, buffers, suspending agents, thickeningagents or viscosity-enhancing agents (such as a hyaluronic acidpolymer). Examples of suitable aqueous and nonaqueous carriers includewater, saline (preferably 0.9%), dextrose in water (preferably 5%),buffers, dimethylsulfoxide, alcohols and polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like). These compositionsmay also contain adjuvants such as wetting agents and emulsifying agentsand dispersing agents. In addition, prolonged absorption of theinjectable pharmaceutical form may be brought about by the inclusion ofagents which delay absorption such as polymers and gelatin. Injectabledepot forms can be made by incorporating the drug into microcapsules ormicrospheres made of biodegradable polymers such aspolylactide-polyglycolide. Examples of other biodegradable polymersinclude poly(orthoesters), poly(glycolic) acid, poly(lactic) acid,polycaprolactone and poly(anhydrides). Depot injectable formulations arealso prepared by entrapping the drug in liposomes (composed of the usualingredients, such as dipalmitoyl phosphatidylcholine) or microemulsionswhich are compatible with eye tissue. Depending on the ratio of drug topolymer or lipid, the nature of the particular polymer or lipidcomponents, the type of liposome employed, and whether the microcapsulesor microspheres are coated or uncoated, the rate of drug release frommicrocapsules, microspheres and liposomes can be controlled.

The compounds of the invention can also be administered surgically as anocular implant. For instance, a reservoir container having a diffusiblewall of polyvinyl alcohol or polyvinyl acetate and containing a compoundor compounds of the invention can be implanted in or on the sclera. Asanother example, a compound or compounds of the invention can beincorporated into a polymeric matrix made of a polymer, such aspolycaprolactone, poly(glycolic) acid, poly(lactic) acid,poly(anhydride), or a lipid, such as sebacic acid, and may be implantedon the sclera or in the eye. This is usually accomplished with theanimal receiving a topical or local anesthetic and using a smallincision made behind the cornea. The matrix is then inserted through theincision and sutured to the sclera.

The compounds of the invention can also be administered topically to theeye, and a preferred embodiment of the invention is a topicalpharmaceutical composition suitable for application to the eye. Topicalpharmaceutical compositions suitable for application to the eye includesolutions, suspensions, dispersions, drops, gels, hydrogels andointments. See, e.g., U.S. Pat. No. 5,407,926 and PCT applications WO2004/058289, WO 01/30337 and WO 01/68053, the complete disclosures ofall of which are incorporated herein by reference.

Topical formulations suitable for application to the eye comprise one ormore compounds of the invention in an aqueous or nonaqueous base. Thetopical formulations can also include absorption enhancers, permeationenhancers, thickening agents, viscosity enhancers, agents for adjustingand/or maintaining the pH, agents to adjust the osmotic pressure,preservatives, surfactants, buffers, salts (preferably sodium chloride),suspending agents, dispersing agents, solubilizing agents, stabilizersand/or tonicity agents. Topical formulations suitable for application tothe eye will preferably comprise an absorption or permeation enhancer topromote absorption or permeation of the compound or compounds of theinvention into the eye and/or a thickening agent or viscosity enhancerthat is capable of increasing the residence time of a compound orcompounds of the invention in the eye. See PCT applications WO2004/058289, WO 01/30337 and WO 01/68053. Exemplaryabsorption/permeation enhancers include methysulfonylmethane, alone orin combination with dimethylsulfoxide, carboxylic acids and surfactants.Exemplary thickening agents and viscosity enhancers include dextrans,polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels,Gelrite®, cellulosic polymers (such as hydroxypropyl methylcellulose),carboxyl-containing polymers (such as polymers or copolymers of acrylicacid), polyvinyl alcohol and hyaluronic acid or a salt thereof.

Liquid dosage forms (e.g., solutions, suspensions, dispersions anddrops) suitable for treatment of the eye can be prepared, for example,by dissolving, dispersing, suspending, etc. a compound or compounds ofthe invention in a vehicle, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol and the like, to form a solution, dispersionor suspension. If desired, the pharmaceutical formulation may alsocontain minor amounts of non-toxic auxillary substances, such as wettingor emulsifying agents, pH buffering agents and the like, for examplesodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate, etc.

Aqueous solutions and suspensions suitable for treatment of the eye caninclude, in addition to a compound or compounds of the invention,preservatives, surfactants, buffers, salts (preferably sodium chloride),tonicity agents and water. If suspensions are used, the particle sizesshould be less than 10 μm to minimize eye irritation. If solutions orsuspensions are used, the amount delivered to the eye should not exceed50 μl to avoid excessive spillage from the eye.

Colloidal suspensions suitable for treatment of the eye are generallyformed from microparticles (i.e., microspheres, nanospheres,microcapsules or nanocapsules, where microspheres and nanospheres aregenerally monolithic particles of a polymer matrix in which theformulation is trapped, adsorbed, or otherwise contained, while withmicrocapsules and nanocapsules the formulation is actuallyencapsulated). The upper limit for the size of these microparticles isabout 5μ, to about 10μ.

Ophthalmic ointments suitable for treatment of the eye include acompound or compounds of the invention in an appropriate base, such asmineral oil, liquid lanolin, white petrolatum, a combination of two orall three of the foregoing, or polyethylene-mineral oil gel. Apreservative may optionally be included.

Ophthalmic gels suitable for treatment of the eye include a compound orcompounds of the invention suspended in a hydrophilic base, such asCarpobol-940 or a combination of ethanol, water and propylene glycol(e.g., in a ratio of 40:40:20). A gelling agent, such ashydroxylethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose or ammoniated glycyrrhizinate, is used. Apreservative and/or a tonicity agent may optionally be included.

Hydrogels suitable for treatment of the eye are formed by incorporationof a swellable, gel-forming polymer, such as those listed above asthickening agents or viscosity enhancers, except that a formulationreferred to in the art as a “hydrogel” typically has a higher viscositythan a formulation referred to as a “thickened” solution or suspension.In contrast to such preformed hydrogels, a formulation may also beprepared so to form a hydrogel in situ following application to the eye.Such gels are liquid at room temperature but gel at higher temperatures(and thus are termed “thermoreversible” hydrogels), such as when placedin contact with body fluids. Biocompatible polymers that impart thisproperty include acrylic acid polymers and copolymers,N-isopropylacrylamide derivatives and ABA block copolymers of ethyleneoxide and propylene oxide (conventionally referred to as “poloxamers”and available under the Pluronic® tradename from BASF-Wayndotte).

Preferred dispersions are liposomal, in which case the formulation isenclosed within liposomes (microscopic vesicles composed of alternatingaqueous compartments and lipid bilayers).

Eye drops can be formulated with an aqueous or nonaqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Drops can be delivered by means of a simple eyedropper-capped bottle or by means of a plastic bottle adapted to deliverliquid contents dropwise by means of a specially shaped closure.

The compounds of the invention can also be applied topically by means ofdrug-impregnated solid carrier that is inserted into the eye. Drugrelease is generally effected by dissolution or bioerosion of thepolymer, osmosis, or combinations thereof. Several matrix-type deliverysystems can be used. Such systems include hydrophilic soft contactlenses impregnated or soaked with the desired compound of the invention,as well as biodegradable or soluble devices that need not be removedafter placement in the eye. These soluble ocular inserts can be composedof any degradable substance that can be tolerated by the eye and that iscompatible with the compound of the invention that is to beadministered. Such substances include, but are not limited to,poly(vinyl alcohol), polymers and copolymers of polyacrylamide,ethylacrylate and vinylpyrrolidone, as well as cross-linked polypeptidesor polysaccharides, such as chitin.

Dosage forms for the other types of topical administration (i.e., not tothe eye) or for transdermal administration of compounds of the inventioninclude powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches, drops and inhalants. The active ingredient may bemixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to theactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Powders and sprays can contain, in additionto the active ingredient, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder ormixtures of these substances. Sprays can additionally contain customarypropellants such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane. Transdermal patches have theadded advantage of providing controlled delivery of compounds of theinvention to the body. Such dosage forms can be made by dissolving,dispersing or otherwise incorporating one or more compounds of theinvention in a proper medium, such as an elastomeric matrix material.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate of such flux can be controlled byeither providing a rate-controlling membrane or dispersing the compoundin a polymer matrix or gel. A drug-impregnated solid carrier (e.g., adressing) can also be used for topical administration.

Pharmaceutical formulations include those suitable for administration byinhalation or insufflation or for nasal administration. Foradministration to the upper (nasal) or lower respiratory tract byinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof one or more compounds of the invention and a suitable powder base,such as lactose or starch. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges, or, e.g.,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, compounds of the invention may beadministered by means of nose drops or a liquid spray, such as by meansof a plastic bottle atomizer or metered-dose inhaler. Liquid sprays areconveniently delivered from pressurized packs. Typical of atomizers arethe Mistometer (Wintrop) and Medihaler (Riker).

Nose drops may be formulated with an aqueous or nonaqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Drops can be delivered by means of a simple eyedropper-capped bottle or by means of a plastic bottle adapted to deliverliquid contents dropwise by means of a specially shaped closure.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents. Also, drug-coated stents may be used.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

A diketopiperazine of formula I, a prodrug of a diketopiperazine offormula I or a pharmaceutically-acceptable salt of either one of them,may be given alone in the methods of the invention. Alternatively, thediketopiperazine, prodrug or salt may be given in combination with eachother and/or in combination with one or more other treatments or drugssuitable for treating the disease or condition. For instance, thediketopiperazine, the prodrug or the salt can be administered prior to,in conjunction with (including simultaneously with), or after anothertreatment or drug. In the case of another drug, the drug and thediketopiperazine, prodrug or salt, may be administered in separatepharmaceutical compositions or as part of the same pharmaceuticalcomposition.

The invention also provides kits. The kit comprises a container holdinga diketopiperazine of formula I, a prodrug thereof or apharmaceutically-acceptable salt of either of them. The kit may furthercomprise one or more additional containers each holding one or moreother drugs suitable for use in the methods of the invention. Suitablecontainers include vials, bottles (including a bottle with a dropper ora squeeze bottle), blister packs, inhalers, jars, nebulizers, packets(e.g., made of foil, plastic, paper, cellophane or another material),syringes and tubes. The kit will also contain instructions foradministration of the diketopiperazine, prodrug or salt and, optionally,the one or more other drugs suitable for use in the methods of theinvention. The instructions may, for instance, be printed on thepackaging holding the container(s), may be printed on a label attachedto the kit or the container(s), or may be printed on a separate sheet ofpaper that is included in or with the kit. The packaging holding thecontainer(s) may be, for instance, a box, or the container(s) maywrapped in, for instance, plastic shrink wrap. The kit may also containother materials which are known in the art and which may be desirablefrom a commercial and user standpoint. The kit may, for instance,contain diet and/or exercise information.

As used herein, “obese” or “obesity” means a medical condition in whichexcess body fat has accumulated to the extent that it may have anadverse effect on health, leading to reduced life expectancy and/orincreased health problems. Obesity increases the likelihood of variousdiseases, particularly heart disease, type 2 diabetes, breathingdifficulties during sleep, certain types of cancer and osteoarthritis.According to the Centers For Disease Control And Prevention (CDC), abody mass index (BMI) of 30 or higher defines adult humans as obese, anda BMI of from 25.0 to 29.9 defines adult humans as overweight. BMI iscalculated by dividing the subject's mass by the square of his or herheight (e.g., (pounds×703)/inches²). “Obesity” in cats and dogs isdefined as a body weight that is greater than 20% above optimal bodyweight.

“Treat,” “treating” or “treatment” is used herein to mean to reduce(wholly or partially) the symptoms, duration or severity of a disease orcondition, including curing the disease, or to prevent the disease orcondition.

“Inhibiting, “inhibit” and similar terms are used herein to mean toreduce, delay or prevent.

As used herein, “a” or “an” means one or more.

As used herein, “comprises” and “comprising” include within their scopeall narrower terms, such as “consisting essentially of” and “consistingof” as alternative embodiments of the present invention characterizedherein by “comprises” or “comprising”. In regard to use of “consistingessentially of”, this phrase limits the scope of a claim to thespecified steps and materials and those that do not materially affectthe basic and novel characteristics of the invention disclosed herein.The basic and novel characteristics of the invention can be treatment ofmetabolic syndrome, suppression of the appetite of an animal in needthereof, treatment of obesity in an animal, reduction of the weight ofan animal in need thereof, reduction of a blood lipid level in an animalin need thereof, treatment of non-alcoholic steatohepatitis in ananimal, inhibition of adipogenesis in an animal, or a combination of anyof the foregoing.

Additional objects, advantages and novel features of the presentinvention will become apparent to those skilled in the art byconsideration of the following non-limiting examples.

EXAMPLES Example 1 Protocol:

-   -   1. Two human male volunteers and one human female volunteer        (ages 61, 50 and 30 respectively) were administered 100 mg of        DA-DKP (packed in a gelatin capsule) orally on an empty stomach        after overnight fasting. The DA-DKP was composed of L-aspartic        acid and L-alanine.    -   2. Venous blood samples were drawn into heparinized and EDTA        vacutainers at baseline (time zero), 2 hours (hrs), 4 hrs and 24        hrs after the ingestion of the DA-DKP.    -   3. Blood samples were analyzed for a complete blood count (CBC)        and a biochemical survey, including plasma lipids at baseline        and at the 24 hrs time points. Plasma was also used for        measurement of the level of DA-DKP by a liquid chromatography        mass spectrometer (LCMS) method (protocol and results given        below).    -   4. Peripheral blood monocytes (PBMC) from heparinized bloods        were extracted by Ficoll gradient method and analyzed by flow        cytometry. The protocol and results are given below.    -   5. Urines were collected for 24 hrs and volumes were recorded.        DA-DKP levels were also measured by LCMS.    -   6. Saliva at 2 hrs was tested for the presence of DA-DKP by        LCMS.

Summary Of Results:

-   -   1. CBC's were unchanged and remained within the normal range.    -   2. BUN, glucose electrolytes, creatinine, liver function tests,        total protein, albumin and calcium were unchanged and within the        normal range at 24 hours post ingestion.    -   3. Lipids decreased across the board for all three subjects at        24 hours post ingestion (see Table 1).    -   4. DA-DKP plasma levels peaked at about 2 hrs and it was not        detectable at 24 hrs post ingestion (see Table 2).    -   5. Urine levels of DA-DKP were calculated (see Table 2).    -   6. Saliva DA-DKP levels were calculated (see Table 2)    -   7. PBMC results are described below.    -   8. Effects noticed by subjects:        -   a. Significant loss of appetite (lasting 8-9 hrs)—3/3            subjects        -   b. Slight dry mouth—2/3 subjects        -   c. Thick saliva—1/3 subjects        -   d. Analgesic effect—1/3 subjects (others did not have pain)*        -   e. Laxative effect—1/3 subjects        -   f. Slight drowsiness—1/3 subjects

* Analgesic Effect:

One subject (61 years old) had left knee and left Sacroiliac (SI) jointpain. Approximately 1-2 hrs after ingestion of the DA-DKP, the knee painwas resolved completely and the SI joint pain was reduced by about 50%.The pain originates probably from inflammation caused by sarcoidosis andwas 6-7 on a scale of 1-10 before treatment. The effect lasted for about4 hrs.

TABLE 1 (% Change in Lipids after 24 Hours) Cholesterol LDL HDLTriglyceride 1 (61 years) −9.2%  −10%  −12%  −2.1% 2 (30 years) −3.0%−7.9%   −7%  +0.5% 3 (50 years)* −1.3% −6.6% −6.7% +28.1% (had largefatty meal the night before) *The 50 year-old male has Gilbert disease

DA-DKP LCMS Measurement

Plasma or urines were passed through a <5 Kd filter and 50 μl of theflow-through fractions were injected into high performance liquidchromatography (HPLC, 2975 system, Waters, Mass.) coupled to a massspectrometer (LCT-TOF, Micromass, UK) and DA-DKP was quantified using astrong anion exchange column (Spherisorb, S5 SAX 250 mm×4.0 mm, Waters)and a ternary mobile phase consisting of water, methanol and 200 mMAmmonium Formate (Sigma Aldrich, Mo.), pH 5.4, at 0.5 ml/min flow ratein a gradient as shown in Table A.

TABLE A 200 mM Ammonium Time (min) Water (%) Methanol (%) Formate pH 5.4(%) 0 25 40 35 10 10 40 50 15 10 40 50 15.01 25 40 35 20 25 40 35

The output of the HPLC was split 1:20 (v:v) and injected into the massspectrometer using negative electrospray ionization (−ESI MS) with ascan range of 80 to 1000 m/z, cone voltage of 30 eV, source temperatureof 100° C., and gas temperature of 300° C. DA-DKP was measured bymonitoring the mass 185 in time, which corresponds to DA-DKP minus asingle proton (—H+). DA-DKP elutes at 10.4 minutes and was quantified byintegrating the area under the curve. The area was compared to astandard curve derived from synthetic DA-DKP standards (DMI Synthesis,Newport Wales, UK) of known concentrations (2000 ng/ml, 1000 ng/ml, 400ng/ml, 200 ng/ml, 100 ng/ml, 20 ng/ml). The calibration curve was foundto fit very well in this range using a quadratic curve on log axes withan R² of 0.997.

The results are presented in Table 2 below. The results are for plasmaunless otherwise noted in Table 2.

TABLE 2 (DA-DKP in μM) 0 15 min 30 min 60 min 120 min 240 min 360 min 24hrs Urine 24 hrs Saliva 2 hrs 1 (61 0 0.14 0.35 0.84 1.68 NA 0.61 0.256.98 NA years (2150 cc) old) 2 (30 0.07 NA NA NA 0.4 0.34 NA 0.14 3.520.29 years (3500 cc) old) 3 (50 0.1 NA NA NA 0.54 0.25 NA 0.09 4.25 0.22years (2000 cc) old)

PBMC Protocol and Results

Peripheral blood mononuclear cells (PBMC) were isolated from peripheralblood, and 2,000,000 cells were cultured in DMEM medium with 10% humanAB serum plus 2% (final concentration) of anti-T-cell receptor complexantibody OKT3. A separate culture without OKT3 antibody was used as theNIL control. After culture overnight at 37° C. cells were washed,incubated with an anti-CD69 antibody, fixed and analyzed on the flowcytometer for CD69 expression. CD69 is an early activation antigenindicative of leukocyte immunocompetence. Results (for a singleindividual) indicated that CD69 expression on CD45RA and CD45RO cellswas decreased after ingestion of DA-DKP from that before DA-DKP intake(time zero) with the greatest effect seen at 4 hours after DA-DKPingestion. Dual histogram analysis indicated that the major effect wasseen on the CD45RO (or previously activated “memory”) subset of T-cells(4 hours, OKT3 Stim. panel).

2. Discussion

The main effects observed after oral administration of DA-DKP were thelack of appetite (which could lead to weight loss, obesity being acardinal feature of the metabolic syndrome) and the decrease in bloodcholesterol, triglycerides, LDL and HDL (dyslipidemia being anotherfeature of the metabolic syndrome). Also, the prevention of activationof previously activated T cells in response to an anti-CD3 stimulation,but not of the naïve T cells, is an important finding (inflammationbeing implicated in the metabolic syndrome). These together with invitro data showing the inhibition of the differentiation ofpreadipocytes into adipocytes by DA-DKP (see Example 2) and thebeneficial effects of DA-DKP on the vascular endothelial barrier (seeco-pending provisional application No. 61/380,404, filed Sep. 7, 2010),support the idea of using DA-DKP as a therapeutic for the metabolicsyndrome.

Example 2 A. Introduction

Adipogenesis is a multi-step process that involves the conversion ofstem cells into fat-storing cells termed adipocytes. Adipose tissue massenlarges and ultimately obesity ensues when energy intake chronicallyexceeds energy expenditure (1) (the numbers in parentheses refer to thereferences listed at the end of this Example 2). At the cellular level,this balance shift manifests itself through mature adipocytesaccommodating excess energy through enhanced triacylglycerol storage(2). When a critical size threshold is reached, it is hypothesized thatoverloaded adipocytes signal preadipocytes to differentiate into newadipocytes to store the excess energy (3). Not surprisingly, obeseanimals have an increased amount of, and larger, adipocytes comparedwith lean animals (4). Any factor that inhibits cellular differentiationinto the adipocyte lineage could be medically useful in treatingobesity. However, a complete understanding of the adipogenic pathway,both inhibitory and stimulatory pathways, is necessary since themodulation of adipose tissue mass may have both advantageous anddeleterious health effects (5).

Recently, due to increased obesity research, adipose tissue is nowrecognized as an endocrine organ due to its hormonal role in theregulation of metabolism, energy intake, and fat storage (6). Adiposetissue accomplishes fat regulation by secreting >50 proteins, termedadipokines, that act in an autocrine, paracrine, and endocrine fashionto control various metabolic functions (6). The most studied adipokinesare adiponectin and leptin which decrease serum free fatty acid,glucose, and triacylglycerol concentrations by insulin sensitivityenhancement (7), and influence food intake by the hypothalamus (8),respectively. Additionally, pro-inflammatory cytokines such as TNFα andIL-6 play an important role in normal and pathological adipogenesis (2).After receptor binding by adipokines and/or cytokines, variousintercellular molecular pathways important to adipogenesis can becomeactivated or inhibited. Activation of mitogen activated protein kinases(MAPK) has an important role in adipocyte differentiation, andderegulation of MAPK can contribute to obesity (9).

B. Materials and Methods

Cell Culture

Subcutaneous preadipocytes (Zen-Bio, Inc., Research Triangle Park, N.C.)were cultured to near confluency at 5% CO₂, 37° C. in PreadipocyteGrowth Medium-2 (PGM-2, Lonza, Walkersville, Md.) containing 10% fetalbovine serum (FBS), L-glutamine, and GA-1000. Cells were harvested usingtrypsin/EDTA (Lonza) and initially seeded in 96-well black/clear bottomplates at 3,000 cells/well. Cells were allowed to form a confluentmonolayer for 7 days in culture.

Cell Dosing and Differentiation 7-day old medium was aspirated and cellswere dosed with DA-DKP (doses used in PGM-2 media: 25, 50, or 100 μM) intriplicate on two plates. The DA-DKP was composed of L-aspartic acid andL-alanine. PGM-2 differentiation media (containing insulin,dexamethasone, indomethacin, and isobutyl-methylxanthine; Lonza) wasadded to one plate, while PGM-2 media was added to the other. Plateswere then placed back in culture and allowed to incubate for 14 days.

Adipogenesis Assay

After the 14 day incubation period, culture media were collected fromall wells and stored at −20° C. for IL-6 ELISA (see below). Wells wererinsed once with 1×PBS (Lonza). 50 μL M-PER lysis solution (ThermoScientific, Rockford, Ill.) were added to all wells, and complete celllysis was achieved by mixing each well. 150 μL 1×PBS was added to eachwell. 5 μL AdipoRed™ Reagent (Lonza) was added to each well, and theplate was mixed immediately by gentle tapping. After 10 minutes,fluorescence was measured on a plate reader set at λ_(EX)=485 nm andλ_(EM)=572 nm.

IL-6 ELISA

Enzyme-linked immunosorbent assay (ELISA) matched-pair antibodies forinterleukin-6 (IL-6) were obtained from Thermo Scientific (Rockford,Ill.). IL-6 was analyzed by ELISA and performed according to themanufacturer's instructions. A 1:10 dilution of each supernatant wasmeasured in duplicate using an IL-6 standard (Thermo Scientific,Rockford, Ill.).

Data Analysis

The triplicate fluorescent values for each treatment were averaged andsubtracted from the average media only (i.e. no cells) wells. For thedifferentiated wells, a % change was calculated by subtracting theaverage fluorescent value for the differentiated cells from theundifferentiated cells for each treatment group. This result was thensubtracted from the difference between the differentiated andundifferentiated control groups (i.e. cells only with no treatment).Finally, this result was then divided by the difference between thedifferentiated and undifferentiated control groups to give a % change.All data are expressed as ±SD. Statistical significance is reported whenp<0.05 based on the Student t-test (Microsoft Excel).

C. Results

DA-DKP caused an increase in adipogenesis at the highest dose (100 μM)while the lowest dose (25 μM) caused a significant decrease inadipogenesis (see FIG. 1). Interestingly, DA-DKP had no significanteffect on IL-6 levels after the 2 week differentiation period (data notshown).

D. Discussion

Obesity has been described as the epidemic of the 21^(st) century due toan increase in the prevalence of obesity as well as an earlier age ofonset (6). This results in a major public health issue since obesityincreases the risk of several chronic diseases such as diabetes,cardiovascular complications, arthritis, asthma, and certain cancers(10). Naturally, the search for a therapeutic agent that reverses thedeleterious effects of obesity is the subject of a considerable amountof current research efforts. In this study, DA-DKP was effective atpreventing adipogenesis (as measured by total intracellular triglycerideconcentrations).

A potential cellular pathway target of DA-DKP is the family of mitogenactivated protein kinases (MAPK). MAPK are serine/threonine kinase thatinclude extracellular signal-regulated kinases (ERK), c-Junamino-terminal kinases (JNK), and p38. MAPKs are important in regulatingboth normal and pathological adipogenesis (9). Previously, theinhibitory effect of DA-DKP on MAPK activity in T-lymphocytes has beenshown (12). It is possible that DA-DKP inhibits adipogenesis by MAPKinhibition, but specific inhibition of MAPK isoforms such as ERK1 andJNK1 seems promising in preventing pathological adipogenesis withoutinterference to other essential MAPK functions (9).

Another important pathway involved in cellular differentiation includingadipogenesis involves the cytoskeleton. Disruption of the actincytoskeleton, forced cell rounding, microtubule disruption, and stressfiber formation are all observed cytoskeletal changes duringadipogenesis (11). DA-DKP has been shown to stabilize the cytoskeletalnetwork in endothelial cells (see co-pending provisional application No.61/380,404, filed Sep. 7, 2010).

DA-DKP is a naturally occurring molecule produced by an unknownmechanism involving the cleavage of the first two amino acids of humanserum albumin and subsequent cyclization of the cleaved dipeptide (13).Additionally, DA-DKP has strong anti-inflammatory characteristics inimmune cell models (12, 13). Interestingly, the pro-inflammatory pathwayplays an important role in adipogenesis (6). In deregulated adiposetissue seen in obesity and diabetes, both local and systemicinflammation as well as insulin resistance and ectopic lipidaccumulation are observed (14). Therefore, the anti-inflammatorytendencies of DA-DKP could be useful in combating the degree ofinflammation seen in obese patients. However, no effect of DA-DKP onIL-6 levels was seen at the end of the differentiation period,suggesting that DA-DKP does not interfere with the pathway thatregulates IL-6 levels during adipogenesis. This does not rule out thepossibility of using DA-DKP as an anti-inflammatory in already obesepatients.

Recently, a clustering of cardiovascular risk factors that includediabetes, obesity, dyslipidemia, and hypertension have been groupedtogether and termed the metabolic syndrome (15). It has been estimatedthat adults with the metabolic syndrome are twice as likely to developcardiovascular disease compared to those without the syndrome (16).Clearly, decreases in abdominal obesity lead to substantial improvementsin the metabolic risk profile resulting in a reduced risk ofcardiovascular disease (17). Another pathological condition associatedwith the metabolic syndrome is the development of non-alcoholicsteatohepatitis (NASH) in obese patients. NASH is characterized by anaccumulation of lipid molecules in the liver accompanied by severeinflammation, fibrosis, and insulin/leptin resistance (18). Therefore,therapeutics that regulate further adipogenesis and/or decreaseinflammation in adipose tissue could decrease the increasedcardiovascular risk associated with the metabolic syndrome and be aneffective treatment for NASH.

The increased prevalence of obesity in Western countries has lead toincreases in various co-morbidities such as cardiovascular disease,hypertension, and diabetes. Searching for a therapeutic that increasesfat metabolism and/or decreases fat storage has been the goal of manynutritional scientists. Here, evidence is presented of theanti-adipogenic effects of a compound that potentially targets a keyregulator of adipogenesis (MAPK), stabilizes the cytoskeleton, anddecreases inflammation, making it an attractive potential treatment formetabolic syndrome and a treatment to reduce obesity.

/E. References for Example 2

-   1. Spiegelman B M, Flier J S. Obesity and the regulation of energy    balance. Cell 2001; 104:531-543.-   2. Harp J B. New insights into inhibitors of adipogenesis. Curr Opin    Lipidol 2004; 15:303-307.-   3. Prins J B, O'Rahilly S. Regulation of adipose cell number in man.    Clin Sci (Lond) 1997; 92:3-11.-   4. DiGirolamo M, Fine J B, Tagra K, et al. Qualitative regional    differences in adipose tissue growth and cellularity in male Wistar    rats fed ad libitum. Am J Physiol 1998; 274:R1460-1467.-   5. Kahn B B, Flier J S. Obesity and insulin resistance. J Clin    Invest 2000; 106:473-481.-   6. Greenberg A S, Obin M S. Obesity and the role of adipose tissue    in inflammation and metabolism. Am J Clin Nutr 2006; 83:461 S-465S.-   7. Fruebis J, Tsao T S, Javorschi S, et al. Proteolytic cleavage    product of 30-kDa adipocyte complement-related protein increases    fatty acid oxidation in muscle and causes weight loss in mice. Proc    Natl Acad Sci USA 2001; 98:2005-2010.-   8. Lee G H, Proenca R, Montez J M, et al. Abnormal splicing of the    leptin receptor in diabetic mice. Nature 1996; 379:632-635.-   9. Bost F, Aouadi M, Caron L, et al. The role of MAPKs in adipocyte    differentiation and obesity. Biochimie 2005; 87:51-56.-   10. Mokdad A H, Ford E S, Bowman B A, et al. Prevalence of obesity,    diabetes, and obesity-related health risk factors, 2001. Jama 2003;    289:76-79.-   11. Feng T, Szabo E, Dziak E, et al. Cytoskeletal disassembly and    cell rounding promotes adipogenesis from ES cells. Stem Cell Rev    2010; 6:74-85.-   12. Shimonkevitz R, Thomas G, Slone D S, et al. A diketopiperazine    fragment of human serum albumin modulates T-lymphocyte cytokine    production through rap 1. J Trauma 2008; 64:35-41.-   13. Bar-Or D, Thomas G W, Bar-Or R, et al. Commercial human albumin    preparations for clinical use are immunosuppressive in vitro. Crit    Care Med 2006; 34:1707-1712.-   14. Gustafson B, Smith U. Cytokines promote Wnt signaling and    inflammation and impair the normal differentiation and lipid    accumulation in 3T3-L1 preadipocytes. J Biol Chem 2006;    281:9507-9516.-   15. Duvnjak L, Duvnjak M. The metabolic syndrome—an ongoing story. J    Physiol Pharmacol 2009; 60 Suppl 7:19-24.-   16. Grundy S M. Metabolic syndrome pandemic. Arterioscler Thromb    Vasc Biol 2008; 28:629-636.-   17. Despres J P. Is visceral obesity the cause of the metabolic    syndrome? Ann Med 2006; 38:52-63.-   18. Larter C Z, Chitturi S, Heydet D, et al. A fresh look at NASH    pathogenesis. Part 1: the metabolic movers. J Gastroenterol Hepatol    2010; 25:672-690.

1-5. (canceled)
 6. A method of treating non-alcoholic steatohepatitis inan animal comprising administering to the animal an effective amount ofan active ingredient, wherein the active ingredient comprises adiketopiperazine, a prodrug of a diketopiperazine or apharmaceutically-acceptable salt of either of them, wherein thediketopiperazine has the formula:

wherein: R¹ and R², which may be the same or different, each is: (a) aside chain of an amino acid, wherein the amino acid is glycine, alanine,valine, norvaline, α-aminoisobutyric acid, 2,4-diaminobutyric acid,2,3-diaminobutyric acid, leucine, isoleucine, norleucine, serine,homoserine, threonine, aspartic acid, asparagine, glutamic acid,glutamine, lysine, hydroxylysine, histidine, arginine, homoarginine,citrulline, phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,thyroxine, cysteine, homocysteine, methionine, penicillamine orornithine; or (b) R¹ is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and togetherwith the adjacent ring nitrogen forms proline or hydroxyproline and/orR² is —CH₂—CH₂—CH₂— or —CH₂—CH(OH)—CH₂— and together with the adjacentring nitrogen forms proline or hydroxyproline; or (b) a derivative of aside chain of an amino acid, wherein the amino acid is one of thoserecited in (a), and the derivatized side chain has: (i) an —NH₂ groupreplaced by an —NHR³ or —N(R³)₂ group, wherein each R³ may independentlybe a substituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl,aryl, alkylaryl, arylalkyl or heteroaryl; (ii) an —OH group replaced byan —O—PO₃H₂ or —OR³ group, wherein each R³ may independently be asubstituted or unsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl,alkylaryl, arylalkyl or heteroaryl; (iii) a —COOH group replaced by a—COOR³ group, wherein each R³ may independently be a substituted orunsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,arylalkyl or heteroaryl; (iv) a —COOH group replaced by a —CON(R⁴)₂group, wherein each R⁴ may independently be H or a substituted orunsubstituted alkyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl,arylalkyl or heteroaryl; (v) an —SH group replaced by—S—S—CH₂—CH(NH₂)—COOH or —S—S—CH₂—CH₂—CH(NH₂)—COOH; (vi) a —CH₂— groupreplaced by a —CH(NH₂)— or a —CH(OH)— group; (vii) a —CH₃ group replacedby a —CH₂—NH₂ or a —CH₂—OH group; and/or (viii) an H which is attachedto a carbon atom replaced by a halogen; provided, however, that when R¹is the side chain of histidine or a derivative of the side chain ofhistidine, then R² cannot be proline or hydroxyproline, and that when R²is the side chain of histidine or a derivative of the side chain ofhistidine, then R¹ cannot be proline or hydroxyproline.
 7. (canceled) 8.The method of claim 6 wherein R¹ and R², which may be the same ordifferent, each is (x) a side chain of an amino acid, wherein the aminoacid is glycine, alanine, valine, norvaline, α-aminoisobutyric acid,2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine,norleucine, serine, homoserine, threonine, aspartic acid, asparagine,glutamic acid, glutamine, lysine, hydroxylysine, arginine, homoarginine,citrulline, phenylalanine, p-aminophenylalanine, tyrosine, tryptophan,thyroxine, cysteine, homocysteine, methionine, penicillamine orornithine, or (y) a derivative of a side chain of one of these aminoacids, wherein the derivatives are those recited in claim
 6. 9. Themethod of claim 6 wherein R¹ and R², which may be the same or different,each is (x) a side chain of an amino acid, wherein the amino acid isglycine, alanine, valine, norvaline, α-aminoisobutyric acid,2,4-diaminobutyric acid, 2,3-diaminobutyric acid, leucine, isoleucine,norleucine, serine, homoserine, threonine, aspartic acid, asparagine,glutamic acid, glutamine, lysine, hydroxylysine, arginine, homoarginine,citrulline, phenylalanine, p-aminophenylalanine, thyroxine, cysteine,homocysteine, methionine, penicillamine or ornithine, or (y) aderivative of a side chain of one of these amino acids, wherein thederivatives are those recited in claim
 6. 10. The method of claim 6wherein R¹, R² or both is the side chain of aspartic acid or glutamicacid or a derivative of such a side chain wherein the —COOH group isreplaced by a —COOR³ group or a —CON(R⁴)₂ group, wherein R³ and R⁴ aredefined as in claim
 6. 11. The method of claim 10 wherein R¹ is the sidechain of aspartic acid or glutamic acid, and R² is the side chain ofalanine or tyrosine.
 12. The method of claim 11 wherein R¹ is the sidechain of aspartic acid, and R² is the side chain of alanine.
 13. Themethod of claim 6 wherein R¹, R² or both is the side chain of methionineor arginine.
 14. The method of claim 13, wherein R¹ is methionine and R²is arginine.
 15. The method of claim 6 wherein the diketopiperazine,prodrug or pharmaceutically-acceptable salt is administered orally. 16.The method of claim 6 wherein the animal is a human. 17-24. (canceled)